High CD46 Receptor Density Determines Preferential Killing of Tumor Cells by Oncolytic Measles Virus

[CANCER RESEARCH 64, 4919–4926, July 15, 2004] High CD46 Receptor Density Determines Preferential Killing of Tumor Cells by Oncolytic Measles Virus

Bambi D. Anderson,1,2 Takafumi Nakamura,1 Stephen J. Russell,1,3 and Kah-Whye Peng1,2 1Molecular Medicine Program, 2Mayo Clinic Cancer Center, and 3Department of Hematology, Mayo Clinic College of Medicine, Rochester, Minnesota

ABSTRACT fects present in tumor cells and not in normal cells (8). One of the most important defense mechanism a cell has against viral infection is Live attenuated Edmonston B strain of measles virus (MV-Edm) is a induction of IFN-␣/␤ and IFN-inducible proteins, resulting in sup- potent and specific oncolytic agent, but the mechanism underlying its pression of protein synthesis and establishment of an antiviral state (9, tumor selectivity is unknown. The virus causes cytopathic effects (CPEs) of extensive syncytial formation in tumor cells but minimal damage or cell 10). However, viruses have evolved diverse strategies to evade or killing in normal cells. The CPE is dependent on expression of viral antagonize the IFN antiviral response (11). Thus, measles virus (MV) proteins and the presence of CD46, the major cellular receptor of MV- encodes the V and C accessory proteins that block IFN-␣/␤ produc- Edm. Using a virally encoded soluble marker peptide to provide a quan- tion and/or signaling, allowing the virus to replicate in the host cell titative readout of the level of viral gene expression, we determined that (12–15). The mechanism underlying MV-C inhibition of IFN-␣/␤ tumor cells and normal cells expressed comparable levels of viral proteins. signaling remains unclear (15), but the MV-V protein blocks the IFN CD46 mediates virus attachment, entry, and virus-induced cell-to-cell response by inhibiting phosphorylation of signal transducers and fusion. Using engineered cells expressing a range of CD46 densities, we activators of transcription 1 and 2 proteins (13). determined that whereas virus entry increased progressively with CD46 MV enters cells by binding via its hemagglutinin (H) attachment density, cell fusion was minimal at low receptor densities but increased dramatically above a threshold density of CD46 receptors. It is well protein to one of two cellular receptors, CD46 (16, 17) or signaling established that tumor cells express abundant CD46 receptors on their lymphocyte activation molecule. The pathogenic wild-type MV surfaces compared with their normal counterparts. Thus, at low CD46 (which is not selectively oncolytic) uses primarily signaling lympho- densities typical of normal cells, infection occurs, but intercellular fusion cyte activation molecule, expressed on activated T cells, B cells, and is negligible. At higher densities typical of tumor cells, infection leads to monocytes/macrophages, as a receptor (18, 19). In contrast, attenuated extensive cell fusion. Intercellular fusion also results in enhancement of vaccine strains such as MV-Edm use predominantly CD46 (20), viral gene expression through recruitment of neighboring uninfected cells which is ubiquitously expressed (usually at low density) by all human into the syncytium, further amplifying the CPE. Discrimination between cells except erythrocytes (21). In addition, CD46 is required to me- high and low CD46 receptor density provides a compelling basis for the diate intercellular fusion. Virally infected cells expressing the MV oncolytic specificity of MV-Edm and establishes MV-Edm as a promising CD46-targeted cancer therapeutic agent. envelope glycoproteins, hemagglutinin (H) and fusion (F), on their cell surfaces fuse with neighboring cells through CD46 to form multinucleated syncytia, the characteristic CPE of MV-Edm infection. INTRODUCTION CD46, also known as membrane cofactor protein, plays an important role in protecting autologous cells from complement attack by Live attenuated Edmonston B strain of measles virus (MV-Edm) serving as a cofactor for Factor I-mediated inactivation of C3b and has potent and specific oncolytic activity against a variety of human C4b, thus blocking the complement cascade at the C3 activation stage tumors, including lymphoma, multiple myeloma, epithelial ovarian (22, 23). Indeed, CD46 is frequently overexpressed on cancer cells cancer, and glioma (1–6). MVCEA, a recombinant MV-Edm genet- compared with their normal counterparts, possibly as a mechanism for ically modified to express human carcinoembryonic antigen (CEA) as cancer cells to overcome lysis by complement (24). Overexpression of a biologically inert soluble marker for noninvasive monitoring of the CD46 and other membrane complement regulatory proteins, CD55 profiles of viral gene expression, is being tested in a Phase I clinical and CD59, has been documented in leukemias and gastrointestinal, trial for patients with recurrent epithelial ovarian cancer (7). hepatocellular, colorectal, endometrial, cervical, ovarian, breast, renal, MV-Edm is selectively oncolytic, causing extensive syncytium and lung carcinomas and found to limit the therapeutic potential of formation and cell killing in a variety of human tumor cells but monoclonal antibody therapy (3, 24). Overexpressed complement minimal cytopathic damage in nontransformed cells such as normal regulatory proteins have also been studied as potential targets for dermal fibroblasts, ovarian surface epithelium, mesothelial cells from cancer therapy using bispecific antibodies and anti-idiotypic vaccina- the peritoneal cavity, and peripheral blood lymphocytes (2, 3). Until tion (25). now, there have been no published studies addressing the mechanisms CPEs induced in MV-Edm-infected cells are dependent on virus underlying the tumor specificity of MV-Edm. However, elucidation of entry, expression of MV-H and MV-F, and the CD46 cellular recep- these mechanisms will be pivotal to future development of MV-Edm tor. We first quantitated the relative expression levels of viral proteins as a cancer therapeutic agent and may provide clues that can poten- in infected tumor cells versus nontransformed cells and found com- tially increase the efficacy or safety of this agent in the clinic. parable levels of gene expression despite striking differences in CPEs. A number of the oncolytic viruses currently being developed for The logical hypothesis to explain these observations was that there cancer therapy are tumor selective because they exploit genetic de- may be a correlation between CD46 receptor density and the strikingly different CPEs observed in tumor cells versus nontransformed Received 3/11/04; revised 5/4/04; accepted 5/12/04. Grant support: The Oliver S. and Jennie R. Donaldson Charitable Trust (B. D. cells on MV-Edm infection. To define the role of CD46 receptor Anderson), Mayo Foundation, Harold W. Siebens Foundation, George W. Eisenberg density in MV-Edm infection, a panel of Chinese hamster ovary Foundation, and NIH Grants CA100634-01 and HL66958-03P4. (CHO) clones expressing a range of surface densities of CD46 was The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with generated. Rodent cells lack CD46, but if they are engineered to 18 U.S.C. Section 1734 solely to indicate this fact. express human CD46, they become infectable by MV-Edm and are Requests for reprints: Kah-Whye Peng, Molecular Medicine Program, 200 First Street SW, Rochester, MN 55905. Phone: (507) 284-8357; Fax: (507) 284-8388; E-mail: susceptible to the CPEs of MV-Edm (26). Using these CHO-CD46 [email protected]. transfectants, MV-eGFP [a recombinant MV-Edm expressing green 4919

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. HIGH CD46 RECEPTOR DENSITY DETERMINES TUMOR CELL KILL fluorescent protein (GFP)], and an adenoviral vector expressing MV- brief centrifugation step and frozen in liquid nitrogen before storage at Ϫ80°C. H/F proteins, we explored the relationship between CD46 receptor Virus titers were obtained by titration on Vero cells and expressed as 50% density, virus entry, and cell fusion. Virus entry increased progres- tissue culture infectious dose (TCID50)/ml. sively with CD46 receptor density and was quite efficient even at Infection Assays. The panel of human tumor and normal cells was infected ϭ relatively low receptor densities. In contrast, syncytium formation and with MV-CEA (MOI 0.2) for2hat37°C, after which the virus inoculum was removed, and the cells were maintained in standard medium for 48 h. The cell killing were minimal at low CD46 densities but increased rapidly cells were fixed with 0.5% glutaldehyde and stained with 0.2% crystal violet above a threshold CD46 expression level. These findings suggest that solution, and the CPEs were photographed. To quantitate the relative levels of CD46 is an interesting new cancer target and that the differential viral gene expression, the cells were infected with MV-CEA (MOI ϭ 0.4) or expression of CD46 in tumor cells versus normal cells dramatically MV-eGFP (MOI ϭ 0.4) and maintained in the presence of FIP to allow increases the susceptibility of tumor cells to the oncolytic activity of analysis of single infected cells by flow cytometry. Forty-eight h later, the MV-Edm, providing a mechanistic basis for tumor specificity of media were harvested, the number of viable cells per well was counted by MV-Edm. trypan blue exclusion, and the percentage of infected GFP-positive cells was analyzed by flow cytometry. The amount of virally encoded CEA marker MATERIALS AND METHODS peptide in the medium was analyzed by the Mayo Central Clinical Laboratory. CHO or CHO-CD46 cells were plated overnight (1 ϫ 105 cells/well) in a Cell Lines. African green monkey kidney Vero cells and human ovarian 12-well plate and infected the next day with MV-eGFP (MOI ϭ 0.5) or Ad5/35 carcinoma SKOV3ip.1, fibrosarcoma HT1080, and epithelial lung carcinoma (MOI ϭ 100) for2hat37°C. The cells were maintained in 10% fetal bovine A549 cells were maintained in DMEM (BioWhittaker, Walkersville, MD) sup- serum-DMEM (Ad5/35) or in medium containing FIP (80 nM). Forty-eight h plemented with 5% (Vero), 10% (HT1080 and A549), or 20% (SKOV3ip.1) later, the percentage of GFP-positive cells was determined by flow cytometry. heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA). NHDF primary Background transduction of parental CHO cells by MV-eGFP (11.5%) or normal human dermal fibroblasts and CASMC coronary artery smooth muscle Ad5/35 (6.7%) was subtracted from the infection data presented in Fig. 3A. cells were purchased from Cambrex (Walkersville, MD) and maintained in media CHO-CD46 cells were plated as described above and infected with K7Ad as recommended by supplier (Cambrex). CD46 expression levels in the panel of H/Ffor6hat37°C, and the cells were maintained in 10% fetal bovine human cells were determined as described below. serum-DMEM. Forty-eight h later, the cells were fixed and stained with 2% Generation of CHO-CD46 Clones. Clones of CHO cells stably expressing crystal violet. The syncytia sizes were analyzed using NIH Image J software. the C1 isoform of human CD46 were generated by transfection of an expres- To determine the importance of CD46 density for bystander recruitment of sion plasmid encoding CD46 C1 (a kind gift from John Atkinson; University neighboring cells into a H/F-expressing focus, CHO-CD46 clone 5 was in- 3 of Washington) into CHO cells using the calcium phosphate method. CD46- fected with MV-eGFP (MOI ϭ 1.0) and plated overnight (1 ϫ 10 cells/well) 5 expressing CHO clones were selected by limiting dilution using 1.2 mg/ml in the presence of FIP to block fusion. The next day, 1 ϫ 10 uninfected CHO, G418 (Invitrogen). A total of 68 clones were screened for CD46 expression by clone 60 (low CD46), clone 78 (medium CD46), or clone 5 cells (high CD46) flow cytometry using a 1:20-diluted phycoerythrin-labeled anti-CD46 antibody were overlaid on the MV-eGFP-infected CHO-CD46 clone 5 cells, and FIP (clone E4.3; BD Biosciences PharMingen) for1hat4°C. The shift in was removed. Forty-eight h later, the cocultures were fixed, and syncytia sizes fluorescence (FL2) is expressed as mean fluorescence index. Sixteen clones were determined using NIH Image J software. expressing a range of CD46 density were used in this study and maintained in 1 mg/ml G418. RESULTS Generation of a Fiber-Modified Adenovirus Expressing MV-H and MV-F (AdK7 H/F). The K7Ad H/F vector was constructed using an in vitro MV Gene Expression in Tumor Cells and Nontransformed ligation method as described previously (27). H coding sequences were cloned Cells Is Comparable. A panel of human cells (SKOV3ip.1, HT1080, downstream of a human cytomegalovirus immediate early promoter/enhancer A549, NHDF, and CASMC) was infected with MV-eGFP or MV- (P ) in the pHM5 shuttle vector (28). The F gene was cloned downstream CMV IE CEA. Expression of GFP by the infected cells facilitates quantitation of the PCMV IE in the pHM11 shuttle vector (28). The plasmid pSK-Ad5F encoding for the human Ad5 fiber was obtained from a fragment of pAdHM48 of the number of MV-infected cells by flow cytometry. The virally (28) that was digested with Csp45I and XbaI. The DNA sequence encoding the encoded soluble CEA marker peptide provides a convenient and linker region and K7 motif was generated by PCR amplification, with precise way to quantitate and monitor MV gene expression (7). As pSKAd5F as the template. Each generated PCR product was digested with shown in Fig. 1A, MV-Edm induced extensive CPEs of syncytial StuI/BamHI or BamHI/AflII and subcloned into the StuI/AflII sites of the formation in MV-infected tumor cells but minimal damage in non- plasmid by a three-part ligation, resulting in pSK-Ad5F/K7. The Csp45I-XbaI transformed NHDF or CASMC cells even at 6 days postinfection fragment from pSK-Ad5F/K7 was subcloned into the pAdHM48 plasmid, (data not shown). Using MV-eGFP, we determined that the striking resulting in pAdHM48-K7. Expression cassettes were transferred from the difference in CPEs was not due to lack of virus infection or gene pHM5 or pHM11 shuttle vectors into the E1- or E3-deleted regions, respec- expression in these nontransformed cells because, for a given MOI, tively, of the adenoviral vector plasmid pAdHM48-K7. The resulting recom- there were comparable numbers of GFP-positive infected tumor cells binant adenovirus genomes were transfected into 293 cells. Because expression of measles H and F proteins causes cell fusion and is toxic to 293 cells, viruses and normal cells (Fig. 1B). To compare MV viral protein synthesis were rescued in the presence of a fusion inhibitory peptide [80 nM; Z-D-Phe- between tumor cells and nontransformed cells and to precisely deter- L-Phe-Gly-OH (FIP); Bachem, Torrance, CA], which blocks H/F-mediated mine the level of viral gene expression per infected cell, the panel of fusion (29). The resulting recombinant adenoviruses were propagated in 293 cells was infected with MV-eGFP or MV-CEA and maintained in the cells in the presence of FIP peptide and purified by CsCl equilibrium centrif- presence of FIP, which prevents syncytium formation, allowing quan- ugation as described previously (30). Purified virion preparations were dia- titation of the number of infected cells by flow cytometry. It is lyzed against 10 mM PBS and 10% glycerol and stored at Ϫ80°C. Viral particle apparent from Fig. 1C that at day 2 or day 4 postinfection, the amount numbers (particles/ml) were calculated from absorbance measurements at of viral protein synthesis per infected cell is similar between tumor 260 nm. cells and nontransformed cells. Thus, the absence of CPEs in non- MV Production and Titration. MV stocks were generated by infecting transformed cells is not due to a significant difference in viral gene Vero cells with MV-eGFP or MV-CEA at a multiplicity of infection (MOI) of 0.02 for2hat37°C, after which the virus was removed, and cells were expression. We therefore turned our attention to cellular factors that maintained in 5% fetal bovine serum-DMEM at 37°C. When 80–90% of the might cause tumor cells to fuse more readily at a given level of viral cells were in syncytia, the media were removed, and the cells were harvested gene expression. into reduced serum Opti-MEM (Invitrogen) media. Cell-associated virions CD46 was a logical choice because syncytium formation is depend- were released by two freeze-thaw cycles, and cell lysates were clarified by a ent on the presence of the CD46 receptor, and tumor cells are known 4920

Fig. 1. The difference in cytopathic effects in MV-Edm-infected tumor cells and normal cells is not due to a lack of virus infection or viral gene expression in normal cells. A, MV-Edm is selectively oncolytic. A panel of human tumor cell lines (SKOV3ip.1, HT1080, and A549) and primary normal human dermal fibroblasts (NHDF) and coronary artery smooth muscle cells (CASMC) were infected with MV-CEA [multiplicity of infection (MOI) ϭ 0.2] for 2 h and photographed 48 h later. B, the absence of cytopathic effects in NHDF and CASMC cells is not due to lack of viral infection. Cells were infected with MV-eGFP (MOI ϭ 0.4), maintained in the presence of Z-D-Phe-L-Phe-Gly-OH, and analyzed by flow cytometry 48 h (f)or96h(u) later. There were comparable numbers of green fluorescent protein-positive infected cells in the cultures. C, viral protein expression per infected tumor cell and normal cell is comparable. Cells were infected with MV-CEA (MOI ϭ 0.4) or MV-eGFP (MOI ϭ 0.4) and maintained in the presence of Z-D-Phe-L-Phe-Gly-OH. The number of infected cells was quantitated by cell counting and flow cytometry. CEA levels in the media were quantitated and expressed as the amount of CEA produced per infected cell at 48h(f) and 96 h (u). D, tumor cells express higher levels of CD46 receptors on their surfaces compared with normal cells. Cells were stained with anti-CD46 PE antibody (black histogram) or an isotype control (gray histogram) and analyzed by flow cytometry. The number is the ratio of the mean fluorescence index of the black histogram:gray histogram and indicates the CD46 receptor expression levels on the cells. to overexpress CD46 receptors compared with their normal counter- Correlation between the Oncolytic Activity of MV-Edm and parts (3, 24). We therefore measured CD46 expression levels on the CD46 Receptor Density. To define the role of CD46 receptor density surfaces of this panel of cells and determined that CD46 expression in MV-Edm entry and CPEs, we used CHO cells and transfected them levels were considerably higher in SKOV3ip.1, HT1080, and A549 with a vector encoding the C1 isoform of human CD46. CD46- tumor cells than in the NHDF or CASMC cells (Fig. 1D). expressing CHO cells are infectable by MV-Edm and express viral 4921

Table 1 Relative CD46 expression levels on surfaces of the panel of proteins, whereupon they fuse with each other to form syncytia (26). a CHO -CD46 clones A total of 68 clones of CHO-CD46 cells were screened for CD46 Clone designation MFI expression by flow cytometry, and 16 clones expressing a range of CHO 14.97 CD46 densities (Table 1) were selected for the studies presented 75 14.28 64 14.59 below. 61 23.50 To first determine the relationship between CD46 receptor density 60 24.52 41 24.69 and the oncolytic activity of MV-Edm, the panel of CHO-CD46 78 51.53 clones was infected with MV-eGFP (MOI ϭ 0.5 and 5.0), and the 25 74.67 extent of CPEs and cell killing in the infected cultures was evaluated. 21 84.53 42 89.21 CPEs (MOI ϭ 0.5) were quantitated by determining the syncytial 46 93.85 index, which is a multiple of the number of syncytia in the culture and 62 346.1 22 476.3 the average syncytium size (measured as pixels/syncytium using NIH 5 509.2 Image J software). Interestingly, we found that the panel of CHO- 31 562.2 63 1000.4 CD46 clones could be divided into two distinct groups based on their 80 1213.9 syncytial indices (Fig. 2A). CPEs were minimal in clones (clones 75, a CHO, Chinese hamster ovary; MFI, mean fluorescence index. 64, 61, 60, and 41) that express a lower density of CD46 receptors

Fig. 2. The extent of MV-Edm-induced cytopathic effects and cell killing is correlated with CD46 receptor density. A, cells were infected with MV-eGFP [multiplicity of infection (MOI) ϭ 0.5], and the extent of cytopathic effects (syncytial index) was quantitated at 48 h postinfection by counting the number of syncytia in the culture and multiplying it by the average syncytium size (pixels) as determined using NIH Image J software. B, cells were infected with MV-eGFP (MOI ϭ 5.0), and cell viability at 48 h postinfection was quantitated by trypan blue exclusion assay. C, representative photographs of MV-eGFP-infected cells (MOI ϭ 5.0) taken at 48 h postinfection. 4922

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. HIGH CD46 RECEPTOR DENSITY DETERMINES TUMOR CELL KILL than clone 78. In contrast, the syncytial indices increased sharply by 2 logs in clones that express higher levels of CD46 receptors (Fig. 2A). Cell viability at 48 h postinfection (MOI ϭ 5.0) was determined by trypan blue exclusion assay, and in line with the minimal CPEs observed in clones 75, 64, 61, 60, and 41 (Fig. 2A), cell death in these infected cultures was minimal compared with significant cell death in the remaining clones (Fig. 2B). Microscopic examination of the MV- eGFP-infected cultures (MOI ϭ 5.0) shows that despite a good level of infection, most of the infected cells remain as unfused single cells in the low CD46- expressing clones (Fig. 2C). In contrast, there was extensive syncytium formation in clones expressing more than the “threshold number” of CD46 receptors. In clone 80, most of the syncytia in the culture have detached from the plate or were no longer viable (loss of GFP fluorescence). These results demonstrate that MV-Edm-induced CPE correlates closely with CD46 receptor density and that the target cells need to express enough CD46 receptors before significant CPE and cell killing will occur. The Relationship between CD46 Receptor Density, Virus En- try, and Virus-Induced Cell Fusion. To determine whether the dramatic differences in CPEs and cell death between low CD46- and high CD46-expressing CHO clones were modulated predominantly by virus entry or by intercellular fusion, we studied the two processes separately. First, we investigated virus entry in CHO or CHO-CD46 clones that were infected with MV-eGFP (MOI ϭ 0.5) and maintained in medium containing FIP. As shown in Fig. 3A, the number of cells infected by MV-eGFP increased progressively as the cell surface density of CD46 receptors increased. For comparison, the panel of CHO-CD46 cells was infected with another CD46 tropic virus, a GFP-expressing chimeric Ad5 adenoviral vector pseudotyped with Ad35 coat (a kind gift from Dr. Andre Lieber; Ref. 31), and a similar relationship between virus infection and CD46 receptor density was observed (Fig. 3B). To investigate the role of CD46 in intercellular fusion, we used an adenoviral vector to express the H and F proteins in our cell panel. In Fig. 3. MV-Edm entry increases progressively as a function of CD46 receptor density. this way, we were able to exclude differences in MV-Edm entry from A, Chinese hamster ovary-CD46 cells were infected with MV-eGFP (multiplicity of infection ϭ 0.5; n ϭ 2) and maintained in medium containing a fusion-inhibitory peptide the equation and achieve high levels of MV-H and MV-F proteins in to prevent intercellular fusion. The percentage of green fluorescent protein-positive cells the CHO-CD46 clones (28). A type 5 serotype adenoviral vector in the infected cultures was analyzed by flow cytometry at 48 h postinfection and plotted expressing the MV-H and MV-F proteins inserted, respectively, in the against CD46 receptor density. B, a chimeric Ad5/35-GFP vector (Ad5 capsid and Ad35 fiber) was used to infect the panel of Chinese hamster ovary-CD46 cells (multiplicity of E1 and E3 positions (AdH/F) under control of a cytomegalovirus infection ϭ 100; n ϭ 3), and the percentage of green fluorescent protein-positive cells was promoter was used (28). The fiber protein of the vector was also analyzed by flow cytometry 48 h later. E, F, Œ, individual data points. modified to display a seven-lysine (K7) COOH-terminal peptide for enhanced CHO cell transduction. CHO and CHO-CD46 cells were transduced with K7Ad H/F at various MOIs, and 2 days later, the cells CEA gene expression increased in parallel with the number of trans- were fixed and stained with crystal violet for visualization of syncytia duced cells (compare Figs. 5A and 3A). However, in the absence of (Fig. 4A, MOI ϭ 300). At low CD46 receptor densities, syncytium FIP, CEA expression levels were little affected in low CD46-express- formation was minimal and small, but as the number of CD46 recep- ing CHO clones, where there was little intercellular fusion, but were tors per cell increased, syncytia formation became increasingly evi- greatly amplified in CHO clones expressing higher densities of CD46 dent. In clones expressing the highest levels of CD46, large syncytia (Fig. 5B). In these high CD46-expressing clones, there is a large merged with each other, causing extensive destruction of the cell bystander effect resulting from intercellular fusion that recruits neigh- monolayers (Fig. 4A). Syncytia sizes in the cell monolayers boring uninfected receptor-rich cells into each syncytium, leading to (MOI ϭ 30, 50, and 100) were also measured quantitatively using an amplification of the overall level of viral gene expression, which in NIH Image J software (the number of pixels per CHO cell is 7000– turn expands the CPE. 9000) and are shown in Fig. 4B. From Fig. 4, it is evident that To confirm the importance of CD46 density in bystander recruit- extensive cell fusion requires “sufficiently” high levels of CD46 ment of neighboring cells by the MV-H/F-expressing cell, CHO or receptors. CD46-expressing clones (low, medium, and high) were overlaid on a Syncytium Formation Amplifies Viral Gene Expression. The small number of MV-eGFP-infected CHO-CD46 cells (clone 5), and importance of syncytium formation in amplifying viral gene expres- the syncytia sizes (n ϭ 10/clone) were determined using NIH Image sion in the cell culture was evaluated by infecting the panel of clones J software. We determined that bystander recruitment, reflected by with MV-CEA (MOI ϭ 0.5) and maintaining the cells in the presence syncytium size, increases as a function of CD46 density. Average or absence of FIP. In these experiments, CEA concentration in the syncytia sizes (mean ϩ SD ϫ 104) in CHO, clone 60, clone 78, or conditioned medium provides a quantitative measurement of overall clone 5 were 0.22 ϩ 0.37, 0.44 ϩ 0.52, 3.86 ϩ 2.25, and 6.20 ϩ 2.55, level of viral gene expression in the infected culture (7). When the respectively, which corresponds approximately to 4, 8, 77, and 124 infected cells were not allowed to fuse and form syncytia (ϩFIP), cells per syncytium. 4923

Fig. 4. Sufficiently high levels of CD46 receptors are required for extensive cell fusion in MV-Edm-infected cultures; below these levels, cell fusion is minimal. A, photographs of Chinese hamster ovary-CD46 cells transduced by an Ad5 adenoviral vector expressing MV-H/F proteins and displaying a K7 peptide on the Ad fiber (K7Ad5 H/F; multiplicity of infection ϭ 300). The cells were fixed and stained with crystal violet at 48 h posttransduction. Photographs of the Chinese hamster ovary-CD46 clones are ranked in order according to the density of CD46 receptors (shown in parentheses) on the cells. B, plot of MV-H/F-induced syncytium size of the respective clones. Digital photographs of K7Ad5 H/F-transduced cells (multiplicity of infection ϭ 30, 50, and 100) were taken, and syncytia sizes (pixels) were determined using NIH Image J software.

DISCUSSION (e.g., CD46, CD55, and CD59) compared with their normal counterparts (3, 24). To define the role of CD46 receptor density in MV- Attenuated MV-Edm has potent and selective antineoplastic activ- Edm-induced CPEs, we used a panel of CHO cells expressing differ- ity, but the mechanism underlying its oncolytic specificity has not ent levels of CD46 receptors and showed that MV-Edm selectively been previously understood (2, 3). Here, we demonstrate that the fuses cells that express a high density of CD46 receptors. Both virus striking difference in CPEs between three tumor cell lines and two entry and intercellular fusion induced by MV-H and MV-F proteins primary normal cells is not due to lack of virus infection or a large difference in viral gene expression levels. We therefore focused on are dependent on CD46 density. However, syncytium formation is CD46 because it is required for virus entry and cell fusion. We affected much more significantly by the same change in CD46 recep- hypothesized that there may be a correlation between CD46 receptor tor density. Cell fusion also amplifies the spread of the initial infection density and the strikingly different CPEs observed in MV-Edm- event by recruiting neighboring uninfected cells into the syncytium infected tumor cells versus nontransformed cells. Mean fluorescence and usurping their host cell machinery for production of viral proteins indices reflecting CD46 receptor density on the tumor cells were and virus progeny. Indeed, viral gene expression was at least 5-fold about 7–10-fold higher compared with the normal cells. This obser- higher in high CD46-expressing clones if syncytia were allowed to vation is in agreement with the vast literature showing that tumor cells form. The syncytia remain viable for some time, producing viral generally express higher levels of complement regulatory proteins proteins and expanding in size, but they eventually die by apoptosis 4924

been tested. Adenoviruses based on the type 5 (Ad5) serotype (group C) have shown promising oncolytic activity and are being tested in the clinic for antitumor efficacy (40). These group C adenoviruses do not use CD46 but interact with the coxsackie adenovirus receptor for attachment and entry into host cells. Coxsackie adenovirus receptor is often down-regulated and expressed at very low levels on tumor cells, and various strategies are therefore currently under investigation to enhance Ad5 infection of tumor cells by redirecting virus entry through cancer-associated receptors such as integrins and epidermal growth factor receptor (41, 42). In contrast to coxsackie adenovirus receptor, CD46 is frequently expressed at high levels on human tumor cells, and pseudotyping the Ad5 capsid with fibers from group B adenoviruses (e.g., with Ad35) can redirect virus entry through CD46 and enhance infectivity of the chimeric virus on tumor cells. Indeed, we found that Ad5/35 infectivity on the panel of CHO-CD46 cells increased progressively with CD46 receptor density. However, in contrast to Ad35, which exploits CD46 for entry only, MV-Edm uses CD46 to mediate both entry and cell-to-cell fusion, leading to destruction of the MV-H/F-expressing cells if they express high levels of CD46 receptors. This unique relationship between the CPE of cell- to-cell fusion (which greatly enhances bystander killing of the in- Fig. 5. Viral gene expression is amplified by syncytium formation. Carcinoembryonic antigen levels in the culture medium of MV-CEA-infected cells (multiplicity of infec- fected cells) and CD46 expression levels makes MV-Edm or the tion ϭ 0.5) at 48 h postinfection are shown in (A) medium containing 80 nM Z-D-Phe-L- fusogenic MV-H/F proteins appealing for use in cytoreductive cancer Phe-Gly-OH (ϩ FIP)or(B) lacking of Z-D-Phe-L-Phe-Gly-OH (Ϫ FIP). therapy. There are likely to be additional factors contributing to the tumor specificity of attenuated MV besides differences in CD46 receptor (4, 32). Discrimination between high and low densities of CD46 density between tumor cells and normal cells (for example, intrinsic receptors on target cells provides a novel strategy for tumor selectiv- fusogenicity). This difference in membrane fusogenicity would mean ity, in addition to the targeting mechanisms used by other oncolytic that different levels of CD46 would be required to trigger efficient agents, for example, adenovirus discrimination of p53 status, IFN fusion in different cell types. In our model, the CHO clones have sensitivity of vesicular stomatitis virus, or exploitation of the ras- similar fusogenicity, and the only difference was the density of CD46 activated pathways by reovirus and herpes simplex virus (8, 33). receptors. The “threshold” receptor density required for extensive It is well established that CD46 is expressed abundantly on tumor fusion will not necessarily be at the same level for human cells. cells, and this has been interpreted as a possible mechanism by which Clearly, additional studies are needed to investigate a wider panel of they resist lysis by complement. In particular, CD46 expression levels human cells, preferably derived from primary sources, to precisely were found to be considerably higher in malignant ovarian cancer, evaluate the range of CD46 expression levels. The primary innate cervical cancer, breast cancer, endometrial cancer, lung cancer, hep- response of a virally infected cell serves to inhibit viral protein atoma, and leukemia than in corresponding normal tissues (3, 24). synthesis and is coordinated through IFN-␣/␤, double-stranded RNA- However, CD46 has not been previously exploited as a cancer target. dependent protein kinase, 2Ј,5Ј-oligoadenylate synthetase, and the Mx In 2001, Durrant and Spendlove (25) proposed using cancer vaccines proteins (9–11). The IFN-␣/␤ or RNA-dependent protein kinase re- to target CD46 and other membrane regulatory proteins overexpressed sponse pathways are often impaired in tumor cells, but not in normal on tumor cells. They reasoned that the remaining CD46-dim tumor cells, and this is the mechanism underlying the tumor selectivity of a cells would be more highly susceptible to complement-mediated lysis number of RNA viruses currently being tested for cancer therapy, for and therefore easy to eliminate with monoclonal antibody therapy. example, vesicular stomatitis virus (43, 44), reovirus (45, 46), and Anti-idiotypic vaccination or bispecific antibodies that recognize both certain herpes simplex virus mutants (47, 48). If these antiviral mech- tumor antigens and CD55 or CD59 have been tested for cancer anisms are induced by MV-Edm infection, they will serve to amplify therapy, although none that recognize CD46 have yet been tested (24, the difference in selectivity conferred by the role of CD46 density in 25, 34). However, whatever the therapeutic agent may be, it must be regulating CPEs. However, MV-Edm encodes V and C viral proteins able to discriminate the relative receptor density on tumor cells and that can respond to the host defense by antagonizing IFN-␣/␤ pro- normal cells and induce potent damage to the tumor cells (25). Our duction and signaling (11–15). We also determined that viral protein study shows that MV-Edm efficiently discriminates between cells synthesis in infected normal cells was only 2–4-fold lower compared with higher and lower surface densities of CD46 and therefore qual- with infected tumor cells, and the striking difference in CPEs cannot ifies as an appropriate agent to target CD46 on tumor cells. There therefore be explained by a preferential shutdown of viral protein remains a possibility that CD46 expression among the tumor cells synthesis in these normal cells as part of their antiviral response. may be heterogenous due to loss or down-modulation of the receptor In conclusion, we have shown that high CD46 receptor density on on a minority of tumor cell surfaces. However, down-modulation of tumor cells is a key determinant of the oncolytic specificity of atten- CD46 can potentially make these cells more susceptible to comple- uated MV. Whereas virus entry increases progressively with CD46 ment lysis and the effects of monoclonal antibody therapy. density, there is a threshold number of CD46 receptors required for CD46 is used as a receptor by other biological agents besides MV cell-to-cell fusion, which leads to death of all of the cells incorporated such as Streptococcus pyogenes (35), Neisseria gonorrhoeae, and into synyctia. This study establishes attenuated MV-Edm as a targeted Neisseria meningitides (36), human herpesvirus 6 (37), and group B oncolytic agent that can discriminate between high CD46 receptor adenoviruses (31, 38). With the exception of the group B adenoviruses densities typical of tumor cells and low CD46 receptor densities (39), the oncolytic potential (if any) of these pathogens has not yet typical of nontransformed cells. 4925

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Bambi D. Anderson, Takafumi Nakamura, Stephen J. Russell, et al.

Cancer Res 2004;64:4919-4926.

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